Compositions for radiolabeling diethylenetriaminepentaacetic acid (dtpa)-dextran

ABSTRACT

The subject invention relates to the compositions for radiolabeling Diethylenetriaminepentaacetic Acid (DTPA)-dextran with Technetium-99m and for stabilizing the DTPA-dextran Cold Kit. The composition contains Stannous Chloride ions to reduce  99m Tc-pertechnetate, Ascorbic Acid to reduce stannic ions to stannous ions to maintain a reducing environment, α,α-Trehalose to add bulk and to stabilize the lyophilized composition without interfering with the radiochemical yield, and Glycine to transchelate Technetium-99m under highly acidic conditions to facilitate radiolabeling DTPA-dextran with high radiochemical purity. In addition, the invention pertains to methods for making and using the compositions. The reconstitution of the lyophilized composition by  99m Tc-pertechnetate, resulting in radiolabeled  99m Tc-DTPA-dextran in a composition between pH 3 to 4. This invention contains a Diluent vial, which when used will shift the pH to a moderately acidic pH, which would provide less pain on injection and ease-of-use to clinical practioners for adjusting its potency.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/461,306, filed May 1, 2012, now Pat. No. 8,545,808, which is adivisional of U.S. application Ser. No. 12/362,778, filed Jan. 30, 2009,which are hereby incorporated in their entirety herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Not applicable.

BACKGROUND

The present disclosure relates to the field of oncology and moreparticularly to the radiolabeling of a cancer detection agent.

Sentinel node biopsy is rapidly gaining acceptance as a common practicefor melanoma and breast cancer diagnosis (Vera, D. R. et al. (2001) J.Nucl. Med. 42, 951-959). This technique has not been standardized; ittypically involves the use of a ^(99m)Tc-colloid and a blue dye. Theradioisotope, ^(99m)Technetium, that is used in the colloid imagingagent and in the current invention, has several desirable properties:its ready availability, relatively low cost, excellent imaging quality,and its short half-life of 6 hours. This radiotracer is employedpreoperatively to ascertain the location of the sentinel node and, then,it is used intraoperatively to pinpoint the dissection of the sentinelnode(s). The blue dye, which is cleared rapidly through the lymphchannels and nodes, is used to visually confirm the selection of theradioactive node as the sentinel node. Because this biopsy procedurevaries with individual practitioners, it is difficult to trainpractitioners with a consistent skill set and consequently, thesebiopsies result in a wide range of reported false-negative rates (i.e.,0 to 12%—see Vera, D. R. ibid.).

There is another hurdle to standardization of this sentinel node biopsytechnique. There is no blue dye or ^(99m)Tc-labeled agents specificallydesigned for sentinel node detection or extraction. Currently, the FDA(U.S. Food and Drug Administration) has not approved any dye, or^(99m)Tc-labeled agent for sentinel node diagnosis. Thus, the followingradiopharmaceutical agents are used off-label: ^(99m)Tc-sulfur colloid,filtered ^(99m)Tc-sulfur colloid, ^(99m)Tc-antimony trisulfide, andseveral preparations of ^(99m)Tc-labeled albumin microcolloids. (Note:Colloids are non-targeted particles that are sticky.) None of theseagents display ideal properties of rapid injection site clearance orhigh sentinel node extraction (Hoh, C. K., et al. (2003). Nucl. Med.Biol. 30, 457-464).

Hence, the development of a nuclear imaging diagnostic kit that isdesigned to meet the goals of optimal sentinel node detection (i.e.,rapid injection site clearance and low distal lymph node accumulation)is an unmet medical need in breast and melanoma cancer treatment.

BRIEF SUMMARY

The present disclosure provides a composition containing a dextranconjugated with a bifunctional chelating agent, such as, DTPA, with easeof use as an “instant” kit involving a single lyophilized vial and aliquid diluent vial, having high radiochemical purity uponradiolabeling. The present disclosure also provides long-term storagestability, as well as sufficient reconstituted stability to facilitateits pharmaceutical or clinical use for ease of manipulation andadministration as a diagnostic imaging agent.

Upon addition of Sodium ^(99m)Tc-pertechnetate, the present disclosuredisplays high radiochemical purity (i.e., >90% ^(99m)Tc-DTPA-dextranpurity) for the bifunctional ligand, DTPA, which are conjugated to anumber of amino-terminated leashes on to a dextran molecule via an amidebond with one of its five carboxylic arms. While free DTPA undoubtedlycoordinates all five of its deprotonated carboxylic groups to bind toheavy metal ions, such as, for example, ¹¹¹Indium, as a potentialoctadentate ligand (also contains three nitrogen atoms—see H. R. Maecke,et al. (1989) J. Nucl. Med. 30, 1235-1239), the heptadentate DTPA bindswith decreased thermodynamic stability, which makes it more susceptibleto competition for binding ^(99m)Tc ions, possibly resulting indecreased radiochemical purity.

The high radiochemical purity of ^(99m)Tc-DTPA-dextran was achieved bydecreasing the pH to between about 2 and 4, screening for non-competingconstituents and identifying the ideal transchelator, Glycine (whichalso serves as a pH buffer), and utilizing the following facts: (1) thedistribution of competing ligands for ^(99m)Tc is determined byassociation rate constants, and (2) the dissociation rate constants for^(99m)Tc from its DTPA-dextran complex is very slow and pH-dependent.Hence, the high efficiency of radiolabeling DTPA-dextran is enhanced bythe transient binding to Glycine under highly acidic conditions, Glycinetransferring the radioisotope to DTPA-dextran that more avidly binds itand the retention of the Technetium-99m (due to its slow dissociationrate constant) after the pH of this “instant” kit is shifted to mildlyacidic conditions by its diluent.

The present disclosure further provides a phosphate buffered salinediluent, enabling patient comfort by shifting pH from harsh acidicconditions (i.e., pH between about 3 and 4), which would cause pain oninjection, to moderately acidic conditions (i.e., pH>˜5), which would bewell tolerated (M. Stranz and E. S. Kastango (2002) Int. J. Pharm.Compound. 6(3), 216-220).

The present disclosure further provides a reducing agent, such as, forexample, L-ascorbic acid, which further stabilizes a radiolabeledDTPA-dextran preparation containing excess stannous or stannic ions,preventing the formation of Sn-colloids or other radiochemicalimpurities, such as, Sn⁴⁺. The present disclosure yet prevents theoxidative degradation of the drug substance and its constituents and theautoradiolysis of the radiolabeled drug product by containing L-ascorbicacid in the formulation.

Furthermore, the present disclosure further provides a stable andesthetically pleasing environment for the DTPA-dextran in an amorphousdisaccharide lyophilization cake, allowing for quick reconstitution withSodium ^(99m)Tc-pertechnetate and addition with a buffered salinediluent to produce a clear, non-particulate liquid for ease of use. Thepresent disclosure also provides an inert gaseous headspace bybackfilling the lyophilized vials with pharmaceutical-grade nitrogengas, further stabilizing the stannous ions to provide an excess capacityover the storage lifetime of this invention for reducing Sodium^(99m)Tc-pertechnetate (or, ^(99m)TcO₄ ⁻).

The present method, then, is an improved method for generating highradiochemical purity ^(99m)Tc(III) (and possibly, ^(99m)Tc(IV))complexes of DTPA-dextran with a single, lyophilized vial that isfurther reconstituted with pH-buffered Diluent to shift final solutionpH, resulting in a solution that is stable for at least 6 hours and thatfacilitates patient comfort (Russell, C. D. (1980) J. Nucl. Med. 21,354-360; Russell, C. D. and Speiser, A. G. (1982) Int. J. Appl. Radiat.Isot. 33, 903-906). The formulation of the lyophilized cold kit forDTPA-dextran is an “instant” kit, stabilizing the stannous chloridenecessary to reduce Sodium ^(99m)Tc-pertechnetate in a solid whitelyophilized cake under a nitrogen environment, which has long-termstorage stability. This kit generates high radiochemical purity by theSn²⁺ reduction of ^(99m)Tc-pertechnetate under highly acidic conditions,while maintaining the ^(99m)Technetium-DTPA-dextran complex in greaterthan 90% radiochemical yield following dilution with aphosphate-buffered saline solution to shift the reconstituted solutionpH toward neutrality.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the nature and advantages of the presentprocess, composition, and kit, reference should be had to the followingdetailed description taken in connection with the accompanying drawings,in which:

FIG. 1 shows a typical Size Exclusion Chromatography (SEC) elutionprofile for reconstituted ^(99m)Technetium-labeled Lymphoseek®(registered trademark of Neoprobe Corporation, Dublin, Ohio U.S. Pat.No. 6,409,990) ligand drug product (^(99m)Tc-DTPA-mannosyl-dextran);

FIG. 2 displays a typical elution profile for ^(99m)Technetium-labeledDTPA Standard radiolabeled with 10 milliCuries ^(99m)Tc-pertechnetateusing lyophilized Lymphoseek ligand drug product placebo;

FIG. 3 shows that three excipients (Citrate, Mannitol and L-Cysteine) ofthe initial pilot formulation display significant ^(99m)Tc-labeledpeaks;

FIG. 4 displays a comparison of the initial drug product formulationwith liquid drug substance formulation pilots;

FIG. 5 is a stacked SEC radiochemical elution profile for liquidDTPA-mannosyl-dextran drug substance placebo formulation pilotscontaining a sodium phosphate pH buffer and different combinations oftranschelator (Citrate), reducing agents (Ascorbic Acid) and bulkingagents (Polyethylene Glycol (PEG) 8000), as measured by the SECradiochemical purity method;

FIG. 6 is a stacked SEC radiochemical elution profiles for thecorresponding liquid DTPA-mannosyl-dextran drug substance placeboformulation pilots containing a sodium phosphate pH buffer;

FIGS. 7A and 7B are the stacked SEC radiochemical elution profiles forliquid DTPA-mannosyl-dextran drug substance and placebo formulationpilots containing 20 mM sodium acetate buffer (ACE) at pH 4, Tartrateand PEG 8000;

FIG. 8 are stacked SEC radiochemical elution profiles for liquidDTPA-mannosyl-dextran drug substance and placebo formulation pilotscontaining 20 mM sodium acetate buffer at pH 4 and 6;

FIGS. 9A and 9B are screening studies employing a reducing sugar with aprimary amine and a zwitterionic amino acid, i.e., Sodium Glucosamine(GlcNH) and Glycine (Gly);

FIGS. 10A and 10B are studies relating to the range of the excipientsGlycine and Sodium Ascorbate at two final concentrations: for Gly₁ andGly₂, it is 0.5 and 2.0 mg Glycine/mL, respectively; and for AA₁ andAA₂, it is 1.5 and 0.38 mg/mL Sodium Ascorbate, respectively;

FIG. 11 are stacked SEC radiochemical elution profiles for the liquiddrug substance formulation pilots with 20 mM sodium acetate bufferranging from pH 5 to 4 with Glycine, Sodium Ascorbate and α,α-Trehalose;

FIG. 12 are stacked SEC radiochemical elution profiles for the DMD drugsubstance formulations (containing 25 μM DTPA-mannosyl-dextran (0.5mg/mL), pH buffer, 0.5 mg/mL Glycine, 0.5 mg/mL Sodium Ascorbate, 2%(w/v) α,α-Trehalose, 38.5 mm Sodium Chloride and 75 μg/mL SnCL₂.2H₂O)with the addition of 12.5 mCi ^(99m)Tc-pertechnetate. The pH buffer isAcetate, pH 4; Phosphate, pH 3; and Phosphate, pH 2 (from top panel tobottom); and

FIG. 13 displays the stacked SEC radiochemical elution profiles for theDMD drug substance formulations at pH 3, 2 and 4 containing thefollowing excipients: 25 μM DTPA-mannosyl-dextran (0.5 mg/mL), 0.5 mg/mLGlycine, 0.5 mg/mL Sodium Ascorbate, 2% (w/v) α,α-Trehalose and 75 μg/mLSnCL₂.2H₂O (with 10 mM Sodium Acetate at pH 4).

The drawings will be described in greater detail in the examples below.

DETAILED DESCRIPTION

The key to development of a commercial “instant” kit for sentinel nodediagnosis is the rational design of an imaging agent that will possessthe properties required for optimal sentinel node detection. Theseproperties are a small molecular diameter and high receptor affinity,yielding a radiopharmaceutical agent with a rapid injection siteclearance rate and low distal lymph node accumulation (Vera, D. R.ibid.). In the present invention, the drug substance that is employeduses a dextran platform to deliver the radiolabel. The dextran backboneis a pharmaceutical-grade, average molecular-weight polymer of about9,500 that is very hydrophilic, lacking in charge, and flexible. Allthese physical properties reduce migration across membrane walls, whichfacilitate rapid injection site clearance. The dextran polymer isconjugated to amine-terminated tethers that are coupled to DTPA groups,giving the molecule high receptor affinity to complex ^(99m)Technetium.The high signal density of ^(99m)Tc-DTPA-mannosyl-dextran enables betterdetection of the sentinel node(s) due to a higher signal-to-backgroundratio.

The addition of mannosyl groups, which are conjugated to otheramine-terminated tethers, gives the binding specificity toDTPA-mannosyl-dextran to differentiate it from its alternative,non-targeted imaging agents. DTPA-mannosyl-dextran binds avidly tomannose-terminated glycoprotein receptors in vitro (Vera, D. R. ibid.).In rabbit biodistribution studies, it was shown that^(99m)Tc-DTPA-mannosyl-dextran diffuses into lymph channels, flows tothe sentinel node, and binds to mannose-binding glycoprotein receptorsin macrophages and dendritic cells present in the sentinel node (Hoh, C.K. ibid.; Fiete, D. and Baenziger, J. U. (1997) J. Biol. Chem. 272(23),14629-14637; Ramakrishna, V. et al. (2004) J. Immunol. 172, 2845-2852).Thus, ^(99m)Tc-DPTA-mannosyl-dextran is a superior targeted^(99m)Tc-labeled diagnostic agent for sentinel node detection (Hoh, C.K., ibid.). While the pre-clinical and Physician Phase I trials of^(99m)Tc-DTPA-mannosyl-dextran successfully employed a radiolabelingprocedure that used multiple fluid transfers and multiple vials, thisdosing format would have been undesirable for commercial usage.

In order to commercialize this important nuclear imaging agent, thecomposition (formulation) and methods for making this composition ofLymphoseek® ligand drug product have been developed, which is thesubject of the present disclosure. The development of a^(99m)Technetium-labeled nuclear imaging “instant” kit is a delicatecounterbalance between high radiochemical efficiency and the formationof non-specific ^(99m)Tc-labeled materials (i.e., ^(99m)Tc-colloid, or^(99m)Tc-labeled formulation excipients). Also, reduced ^(99m)Technetiumneeds to be prevented from reoxidating to ^(99m)TcO₄ ⁻. Hence, alyophilized formulation was developed to stabilize stannous ions underan inert nitrogen environment.

In this disclosure, the composition accomplishes this delicate balancingact by utilizing a newly identified transchelator, Glycine, under highlyacidic conditions. A transchelator is a weak chelator that transientlybinds reduced ^(99m)Technetium, facilitating the transfer of thisradioisotope to a stronger chelator, or ligand. The ligand for reduced^(99m)Technetium is derivatized diethylenetriaminepentaacetic Acid(DTPA), a heptadentate bifunctional ligand that coupled to the dextranamine-terminated tether by one of its five carboxylic groups. Thisligand is well known to the practitioners of the art. It has beenincorporated into “instant” kits for radiolabeling peptides and proteins(Hansen et al., U.S. Pat. No. 5,328,679; Zamora and Marek, U.S. Pat. No.6,685,912 B2; and Winchell, U.S. Pat. No. 4,364,920) In these USpatents, DTPA is a bifunctional chelator conjugated to peptides andproteins, usually as an anhydride form covalently attached through itscarbon backbone.

These patents describe a spectrum of transchelators known to the art,such as, for example, Citrate, Tartrate, Phosphate, Phosphonate,Glucoheptonate and even, Ascorbic Acid. But, these transchelators arelargely employed in mildly acidic to neutral pH formulations and caninterfere with radiolabeling the active ingredient with high efficiency.The optimal pH for using Ascorbic Acid as a transchelator is from pH 4.5to 6.2 (Liang et al. (1987) Nucl. Med. Biol. 14, 555-562). This stemsfrom the pK_(a) of its carboxylic group, pH 4.10 (CRC: Handbook ofChemistry and Physics, 75^(th) Edition, David R. Lide, Ph.D. (CRC Press,London)). The pK_(a) of the carboxylic group of Glycine is 2.34. Itscarboxylic group remains functional under highly acidic conditions(e.g., partially deprotonated at pH 2 and is fully deprotonated at pH4). At the preferred embodiment in this disclosure, Ascorbic Acid isfully protonated. Thus, the composition reduces the potentialinterference of ascorbic acid, utilizing the beneficial properties ofthis antioxidant, while employing Glycine as an optimal transchelator.

When the covalently coupled DTPA binds reduced ^(99m)Technetium, theprobable principal oxidation state is ^(99m)Tc(III) under acidicconditions, which should result in stable complex with a zero net charge(Russell, C. D. (1980) J. Nucl. Med. 21, 354-360). The liquidcomposition at pH 2 radiolabels DTPA-dextran successfully, shifting to ahigher pH on the addition of the diluent. But, at pH lower than, orequal to 2.7, the DTPA groups are fully protonated, possibly resultingin the total collapse of the lyophilized formulation at pH 2 (Hnatowich,D. J. et al. (1995) J. Nucl. Med. 36, 2306-2314). Hence, the preferredembodiment is to have the composition range from pH about 3 to about 4to enable high radiochemical efficiency, while shifting pH to greaterthan about pH 5 on dilution of the reconstituted “instant” kit withphosphate-buffered saline Diluent, which would be well tolerated bypatients. In this application,all ingredients are desired to beUSP-grade (United States Pharmacopeia). Also, “q.s.” has its standardpharmaceutical meaning of “as much as is sufficient”.

EXAMPLES Example 1 Elution Profiles of ^(99m)Tc-LabeledDTPA-mannosyl-Dextran and DTPA

FIG. 1 shows a typical elution profile for reconstituted^(99m)Technetium-labeled Lymphoseek Ligand Drug Product(^(99m)Tc-DTPA-mannosyl-dextran), Lot NMK001, measured by aradioactivity (Nal, set at 1000 cps/Volt) detector using Size ExclusionChromatography (SEC). The conditions for this SEC radiochemical puritymethod are as follows: a TSKgel column, Tosoh Bioscience, G3000PW_(XL)(7.8×30 cm, 6 μm, with a column temperature of 25±5° C.) is employedwith an isocratic mobile phase of 50 mM phosphate buffer, pH 7.2, and300 mM sodium chloride. The lyophilized vial is reconstituted with 0.8cc of 10 milliCuries of ^(99m)Tc-pertechnetate, mixed, and allowed toradiolabel for at least 10 minutes at ambient room temperature prior topartially neutralizing the sample in 0.2 cc Phosphate-buffered saline. Arefrigerated drug product sample, 15 μL, is injected and run at 0.6mL/minute for a run time of 40 minutes; the retention time of the^(99m)Tc-DTPA-mannosyl-dextran (^(99m)Tc-DMD) peak is about 12 to 12.5minutes, stretching between 9 and 15 minutes with a tailing shoulder of^(99m)Tc-labeled excipients eluting at a radioactive peak of about 15 to15.5 minutes.

The elution profile is very similar to that the potency method using thesame column and mobile phase, employing a Refractive Index detector (dueto the absence of a UV/VIS absorbance). The broad elution peak for^(99m)Tc-DTPA-mannosyl-dextran is a result of the heterogeneity of thedextran polymer, which is further acerbated by the heterogeneity of thecoupling of mannosyl and DTPA groups to amino-terminated leases ondextran (Vera, D. R. et al. (2001) J. Nucl. Med. 42, 951-959). The goalof the DTPA-mannosyl-dextran formulation was to achieve greater than 95%radiochemical purity in the bulk liquid drug substance formulation andgreater than 90% radiochemical purity in the reconstituted lyophilizeddrug product.

FIG. 2 displays a typical elution profile for ^(99m)Technetium-labeledDTPA Standard radiolabeled with 10 milliCuries ^(99m)Tc-pertechnetateusing lyophilized Lymphoseek Ligand Drug Product Placebo (i.e., 4.5 mML-Glycine, pH 3, 2.5 mM Sodium L(+)-Ascorbic Acid, 2% (w/v)α,α-Trehalose and 75 μg/mL Stannous Chloride Dihydrate), measured by aRadioactivity (Nal) Detector using SEC radiochemical purity method,described above. The ^(99m)Tc-DTPA peak retention time is about 15minutes, eluting between 14 and 16 minutes, which are the approximateretention times for almost all of the ^(99m)Tc-labeledlow-molecular-weight excipients (data not shown).

Example 2 Initial Pilot Formulation: Investigating InterferingExcipients

In FIG. 3, the topmost stacked radiochemical elution profile shows theinitial lyophilized formulation pilot (5 μM (0.1 mg/mL)DTPA-mannosyl-dextran, 20 mM Sodium Citrate, pH 5.6, 5.7 mM SodiumL-Cysteine, 2% (w/v) D-Mannitol and 75 μg/mL Stannous Chloride,Dihydrate) reconstituted with 10 milliCuries ^(99m)Tc-pertechnetate andrun via the SEC radiochemical purity method. (The initial lyophilizeddrug product formulation pilot just preceded the development of the SECradiochemical purity method.) This elution profile clearly shows thatthe ^(99m)Tc-DMD peak has less than about 25% radiochemical purity.

The following screening method (in the order of addition) was employedto determine potential interfering excipients in pilot formulations: (1)for drug substance placebo formulations, add 50 μL degassed saline to a1.5 mL plastic test tube with a cap; for drug substance formulations,add 50 μL of 1.2 mg/mL DTPA-mannosyl-dextran in degassed saline for afinal concentration of 0.3 mg/mL DMD; (2) for testing differentexcipients, add 50 μL of a four-fold concentrated, degassed solution;(3) for reduction of ^(99m)Tc-pertechnetate, add 50 μL of 300 μg/mL ofStannous Chloride, Dihydrate in 0.01N degassed Hydrochloric Acid; andimmediately following the addition of SnCL₂, (4) for radio-labeling theformulation with reduced ^(99m)Technetium, add 50 μL of 50 milliCuriesof ^(99m)Tc-pertechnetate for a final concentration of 12.5 mCi^(99m)Tc-pertechnetate. (Note: Solutions were degassed by bubblingnitrogen gas for at least one hour.) Then, mix and let stand at ambienttemperature at least 10 minutes before transferring to a capped HPLCautosampler vial to perform the SEC radiochemical purity assay.

FIG. 3 shows that three excipients of the initial pilot formulationdisplay significant ^(99m)Tc-labeled peaks. Proceeding from the topmoststacked radiochemical elution profile in a downward manner, the secondelution profile shows a substantial ^(99m)Tc-Citrate peak at RT˜14.5min, which may account for a significant amount of the ^(99m)Tc-labeledinterference at RT˜14.5 min in the topmost pattern. Citrate is a knowntranschelator of DTPA at a pH range of 5 to 6 (Hnatowich, D. J., Chapter8, pg. 175, Cancer Imaging with Radiolabeled Antibodies (Goldenberg, D.M., ed., 1990: Kluwer Academic Publishers, Boston/Dordrecht/London)),but it appears to be too strong to use in the current formulation. Inthe third radiochemical elution profile, it appears that D-Mannitolcompetes for ^(99m)Tc, eluting at RT˜16 min. This unexpectedinterference may be due to the impurities present in this naturalproduct. In the fourth and fifth radiochemical elution profiles, twodifferent L-Cysteine concentrations were employed: 0.25 and 1.0 mg/mLL-Cysteine in the final concentration, respectively. While the fourthelution profile displays some interference binding, the fifth elutionprofile at 1 mg/mL L-Cysteine clearly shows that Cysteine binds ^(99m)Tcand interferes with the transchelation of Citrate, eluting at retentiontimes ranging from 21 to 23 minutes. The sixth radiochemical elutionprofile involves the addition of 1 mg/mL sodium L(+)-Ascorbic AcidDihydrate to a Citrate formulation; Ascorbic Acid does not appear tointerfere with ^(99m)Tc-Citrate.

FIG. 4 displays a comparison of the initial drug product formulationwith liquid drug substance formulation pilots. The topmost stackedradiochemical elution profile is the initial drug product formulationand the second profile is that of Sodium Citrate in saline with SnCL₂added to reduce 12.5 mCi ^(99m)Tc-pertechnetate. In the third and fourthradiochemical elution profiles, the DTPA-mannosyl-dextran drug substanceis partially radiolabeled with a significant ^(99m)Tc-Citrate eluting atabout 14.5 minutes. Hence, the use of Sodium Citrate is not a suitablepH buffer\transchelator choice.

Example 3 Screening for pH Buffers, Transchelator and Bulking Excipientsfor Enhanced Radiolabeling of DTPA-mannosyl-dextran

FIG. 5 is a stacked radiochemical elution profile for liquidDTPA-mannosyl-dextran drug substance placebo formulation pilotscontaining a Sodium Phosphate pH buffer and different combinations oftranschelator, reducing agents and bulking agents, as measured by theSEC radiochemical purity method. The topmost stacked radiochemicalelution profile shows a small ^(99m)Tc-labeled interference peak withthe 20 mM Sodium Phosphate buffer at pH 4 and 1.5 mg/mL SodiumAscorbate. The second through the sixth elution profiles shows 20 mMSodium Phosphate, pH 4, 75 μg/mL SnCL₂.2H₂O and 12.5 mCi^(99m)Tc-pertechnetate with the following respective potentialexcipients: 1 mg/mL Sodium Citrate; 1% PEG 8000; 1 mg/mL Sodium Citrateand 1.5 mg/mL Sodium Ascorbate; 1.5 mg/mL Sodium Ascorbate and 1% PEG8000; and 1.5 mg/mL Sodium Ascorbate, 1 mg/mL Sodium Citrate and 1% PEG8000. They all display significant ^(99m)Tc-labeled interference peakseluting as early as RT˜14 minutes for ^(99m)Tc-PEG 8000 to a morelow-molecular-weight retention time of ˜15 minutes for ^(99m)Tc-Citrate.

In FIG. 6, the stacked radiochemical elution profiles for thecorresponding liquid DTPA-mannosyl-dextran drug substance placeboformulation pilots containing a Sodium Phosphate pH buffer are seen. ForDTPA-mannosyl-dextran formulations containing 0.3 mg/mL, or 15 μM DMD inFIG. 6, the stacked radiochemical elution profiles show a littlesignificant radiolabeling of the drug substance. The third profile fromthe top displays background levels of ^(99m)Tc-DMD, indicating thatphosphate and PEG 8000 do not serve as satisfactory transchelators. Atone-fifth its pH buffer strength, Citrate is less efficient inradiolabeling drug substance and still interferes in these formulations.In addition, PEG 8000 apparently interferes with the drug substanceyield with its hydroxyl groups and is unsuitable as a bulking agent.Since Sodium Phosphate is not an ideal pH buffer for lyophilization,alternative generally recognized as safe (GRAS) pH buffers werescreened.

In FIGS. 7A and 7B, the stacked radiochemical elution profile for liquidDTPA-mannosyl-dextran drug substance and placebo formulation pilotscontaining 20 mM Sodium Acetate buffer at pH 4 are interspersed. In FIG.7A, the topmost radiochemical elution profile is the drug substanceformulation with the potential transchelator, Sodium Tartrate at 1.5mg/mL, displaying an enhanced radiolabeling of the drug substance with asignificant interfering peak, ^(99m)Tc-Tartrate (see fourth elutionprofile for corresponding placebo formulation). The second and thirdelution profiles in FIG. 7A show little difference in the presence ofSodium Ascorbate and PEG 8000. In FIG. 7B, the radiochemical elutionprofiles demonstrate that the drug substance formulations for the SodiumAscorbate and PEG 8000 combinations with Tartrate have less efficiencyin radiolabeling the drug substance. Finally, the DTPA Standard has asmall tailing edge shoulder with Sodium Acetate and Sodium Ascorbate atpH 4. Hence, the selection of Sodium Tartrate as potential transchelatorin an Acetate pH buffer is unsatisfactory.

In FIG. 8, the stacked radiochemical elution profile for liquidDTPA-mannosyl-dextran drug substance and placebo formulation pilotscontaining 20 mM Sodium Acetate buffer at pH 4 and 6 are alsointerspersed. The topmost and second radiochemical elution profiles showat pH 6, the presence of 1.5 mg/mL Sodium Ascorbate enhances theradiochemical purity of the drug substance, but the third and fourthprofiles indicate that Ascorbate may contribute to a significant and asmaller interference peak of ^(99m)Tc-Ascorbate at RT˜13.5 and ˜15minutes, respectively. The fifth elution profile demonstrates that theradiochemical purity is pH-sensitive, primarily radiolabeling the drugsubstance at pH 4 in the presence of Sodium Ascorbate. The fifth profilemay contain some interfering material co-eluting with the ^(99m)Tc-DMDpeak, as observed in the slight shoulder of the trailing edge of thedrug substance peak as well as the small ^(99m)Tc-labeled peak at RT˜16minutes.

In FIGS. 9A and 9B, screening studies employing a reducing sugar with aprimary amine and a zwitterionic amino acid, i.e., Sodium Glucosamineand Glycine, were conducted on an educated guess that these excipientswould have some transient interactions with ^(99m)Technetium, becausethis radioisotope forms stable complexes with amine and amide nitrogens,carboxylate oxygens, and thiolate and thioether sulfurs with a strongpreference for thiolate sulfurs (Giblin, M. F. et al. (1998) PNAS USA95, 12814-12818). FIG. 9A displays the liquid DTPA-mannosyl-dextran drugsubstance placebo formulation pilots with 20 mM Sodium Acetate buffer atpH 4 with either 1.5 mg/mL Sodium Glucosamine and Glycine in the absenceand presence of Sodium Ascorbate (1.5 mg/mL). These excipients displaybackground level radioactivity with small ^(99m)Tc-labeled peaks atRT˜15 minutes, except for the third elution profile for just SodiumGlucosamine, which has greater than background radioactivity. In FIG.9B, the stacked radiochemical elution profiles for the liquid drugsubstance formulation pilots with 20 mM Sodium Acetate buffer, pH 4, andeither 1.5 mg/mL Sodium Glucosamine, or Glycine are nearly identical,demonstrating an ability to efficiently radiolabel the ^(99m)Tc-DMD peak(at RT12.4 min) as transchelators. Because Sodium Glucosamine is not aGRAS excipient, it was not pursued in subsequent formulations. Glycinewas identified as a potential, non-interfering transchelator.

Since Glycine and Sodium Ascorbate appeared compatible with enhancedradiochemical purity of the drug substance, the range of theseexcipients was investigated. Glycine and Sodium Ascorbate were evaluatedat two final concentrations: for Gly₁ and Gly₂, it is 0.5 and 2.0 mg/mL,respectively; and for AA₁ and AA₂, it is 1.5 and 0.38 mg/mL,respectively. For the Glycine #1 and 2 drug substance formulations(i.e., 15 μM DTPA-mannosyl-dextran, 20 mM Sodium Acetate, pH 4, 75 μg/mLSnCL₂.2H₂O and 12.5 mCi ^(99m)Tc-pertechnetate), the mean average of tworadiolabeling studies for Gly₁ and Gly₂ are 90.7 and 88.7% ^(99m)Tc-DMD,respectively, as measured by the SEC radiochemical purity method. In thepresence of Gly₁, the mean average of two radiolabeling studies for theAA₁ and AA₂ drug substance formulations are 80.3 and 90.3% ^(99m)Tc-DMDpurity, respectively (see FIGS. 10A and 10B).

The screening for suitable bulking agents for lyophilization wasconducted in 20 mM Sodium Acetate, pH 4 to 5, formulations containingGlycine as a transchelator and Sodium Ascorbate as anantioxidant\reducing agent. It was determined that polymeric excipients,such as, PEG 2000 and Polyvinylpyrrolidone interfered with theefficiency of radiolabeling DTPA-mannosyl-dextran (data not shown).Finally, α,α-Trehalose (2% w/v) was identified as a potentialnon-interfering bulking agent for the liquid drug substance formulation.In FIG. 11, the stacked radiochemical elution profiles for the liquiddrug substance formulation pilots with 20 mM Sodium Acetate bufferranging from pH 5 to 4 with Glycine and Sodium Ascorbate shows that 2%α,α-Trehalose has little, if any, interference in the radiochemicalpurity of ^(99m)Tc-DMD. Furthermore, there is significant pH sensitivityin the Sodium Acetate formulations (15 μM DTPA-mannosyl-dextran, 20 mMSodium Acetate, 1 mg/mL Glycine, 1 mg/mL Sodium Ascorbate, 2% (w/v)α,α-Trehalose, 38.5 mM Sodium Chloride, 75 μg/mL SnCL₂.2H₂O and 12.5 mCi^(99m)Tc-pertechnetate), giving 86.8, 88.4 and 93.8% ^(99m)Tc-DMD purityfor pH 5.0, 4.5 and 4.0, respectively. Hence, the screening process foridentifying suitable excipients for potential use in a lyophilized drugproduct kit formulation was completed. The next step is to optimize theformulation, demonstrate the feasibility of the lyophilized kit formatand develop a reconstitution procedure.

Example 4 Optimizing the Formulation for Enhanced Radiolabeling ofLyophilized DTPA-mannosyl-dextran Drug Product

The apparent pH sensitivity of the Acetate buffer formulations, givingimproved radiochemical purity for ^(99m)Tc-DMD at decreasing pH, neededto be explored. The initial pH studies employed 10 mM Sodium Phosphateat pH 2 and 3 and as a control, 20 mM Sodium Acetate at pH 4. FIG. 12shows the stacked radiochemical elution profiles for the DMD drugsubstance formulations (containing 25 μM DTPA-mannosyl-dextran (0.5mg/mL), pH buffer, 0.5 mg/mL Glycine, 0.5 mg/mL Sodium Ascorbate, 2%(w/v) α,α-Trehalose, 38.5 mM Sodium Chloride and 75 μg/mL SnCL₂.2H₂O)with the addition of 12.5 mCi ^(99m)Tc-pertechnetate. The topmostelution profile displays the Acetate formulation at pH 4, which has96.9% ^(99m)Tc-DMD purity, as measured by the SEC radiochemical puritymethod. This formulation meets our goal for liquid drug substanceformulation (i.e., greater than 95% ^(99m)Tc-DMD purity). Unfortunately,the 10 mM Sodium Phosphate formulations at pH 3 and 2 have substantial^(99m)Tc-labeled interference peaks at RT˜14.0 minutes (see FIG. 12).Thus, it was determined that Glycine/Hydrochloric Acid should serve as asuitable acidic pH buffer as well as a potential non-interferingtranschelator. FIG. 13 displays the stacked radiochemical elutionprofiles for the DMD drug substance formulations at pH 3, 2 and 4containing the following excipients: 25 μM DTPA-mannosyl-dextran (0.5mg/mL), 0.5 mg/mL Glycine, 0.5 mg/mL Sodium Ascorbate, 2% (w/v)α,α-Trehalose and 75 μg/mL SnCL₂.2H₂O (with 10 mM Sodium Acetate at pH4). It was fortuitous to employ Glycine Hydrochloride as an acidic pHbuffer and a transchelator, because the pH 3 and pH 2 drug substanceformulations exhibit 97.6 and 97.1% ^(99m)Tc-DMD purity, respectively,and meeting the desired goal of the drug substance formulation (see FIG.13). In FIG. 13, the Acetate formulation at pH 4 failed to meet the goalfor drug substance formulation (93.6% versus >95% ^(99m)Tc-DMD purity),but this may due to day-to-day variability in the preparation of theformulation, incomplete degassing of the solutions, the inadequatemixing of the Stannous Chloride Dihydrate, etc. The GlycineHydrochloride buffer may be utilized in pH 4 formulations in addition tothe Acetate buffer.

The Class I glass vials were filled with sterile-filtered aliquots ofthis pH study, 1.05 mL, into 3 mL vials. Stoppers were placed in thenecks of these vials, and the vials were placed on the VirTisLyophilizer shelves for lyophilization. After the lyophilization cyclewas completed, the vials were backfilled with nitrogen gas andstoppered. Subsequently, the stoppered vials were crimped with aluminumseals. On visual inspection, the lyophilized cakes for the Acetate, pH4, and the Glycine, pH 3, drug product formulation vials retained theiramorphous structure and appeared to have dried to low residual moisture.In contrast, the Glycine, pH 2, drug product formulation vials weretotally collapsed (i.e., devoid of structure). The preferred embodimentof this invention is the pH 3 drug product formulation (i.e., 12.5 to 25μM DTPA-mannosyl-dextran (0.25 to 0.5 mg/mL), 0.5 mg/mL Glycine, pH 3,0.5 mg/mL Sodium Ascorbate, 2% (w/v) α,α-Trehalose and 75 μg/mLSnCL₂.2H₂O).

Example 5 Developing the Reconstitution Procedure, Including Using aPhosphate-Buffered Saline Diluent, for Improved Ease-of-Use inRadiolabeling Lyophilized DTPA-mannosyl-dextran Drug Product

Due the final pH of the Lymphoseek Ligand Drug Product formulation,about pH 3, which is lower than the recommended pH for parenteral drugs(Stranz, M. and Kastango, E. S. (2002) Int. J. Pharm. Compound. 6(3),216-220), it was decided to utilize a Diluent that neutralizes pHfollowing the reconstitution with ^(99m)Tc-pertechnetate to a lesspainful and harmless pH (e.g., between pH 5 and 9). Sodium^(99m)Tc-pertechnetate is eluted from a Molybdenum-99 generator with0.9% Sodium Chloride, or isotonic saline. The Lymphoseek Ligand DrugProduct is formulated to meet the recommendations of the InfusionNursing Society to be less than 500 mOsm\L following reconstitution with1 mL of Sodium ^(99m)Tc-pertechnetate. A suitable Diluent was identifiedfor use with human parenterals, Buffered Saline for Injection from GreerLaboratories. The formulation of this Diluent is: 0.107% SodiumPhosphate, Heptahydrate, 0.036% Potassium Phosphate (desirably USP—NF,United States Pharmacopeia—National Formulary), 0.5% Sodium Chloride and0.4% Phenol. It is recommended that the lyophilized Lymphoseek LigandDrug Product vial is reconstituted with 0.7 cc of 10 to 50 mCi of Sodium^(99m)Tc-pertechnetate for at least 10 minutes at ambient roomtemperature, mixed intermittently and then, diluted with 0.3 cc ofBuffered Saline for Injection. The Lymphoseek® Ligand Drug Product hasat least twelve hours of reconstituted stability, but it is recommendedthat the reconstituted drug product be administered within six hours(data not shown). Hence, the neutralized ^(99m)Tc-labeled LymphoseekLigand Drug Product should be well tolerated by patients uponintradermal injection.

While the process, composition, and kit have been described withreference to various embodiments, those skilled in the art willunderstand that various changes may be made and equivalents may besubstituted for elements thereof without departing from the scope andessence of the disclosure. Additionally, many modifications may be madeto adapt a particular situation or material to the teachings of thedisclosure without departing from the essential scope thereof.Therefore, it is intended that the disclosure may not be limited to theparticular embodiments disclosed, but that the disclosure will includeall embodiments falling within the scope of the appended claims. In thisapplication the US measurement system is used, unless otherwiseexpressly indicated. Also, all citations referred to herein areexpressly incorporated herein by reference.

1. A composition for radiolabeling diethylenetriaminepentaacetic Acid(DTPA)-dextran with Technetium-99m, compromising: (a) a DTPA-dextranwith a concentration of up to 0.50 mg/vial; (b) a sugar selected fromthe group of non-reducing disaccharides with a concentration up to 2%(w/v); (c) a non-sulfhydryl anti-oxidant wherein the concentration is inthe range of about 0.5 mg/vial; (d) a stannous salt wherein theconcentration of the dihydrate form of the stannous salt is up to 75micrograms/vial; (e) a pH buffer selected from a group of pH buffers inthe concentration range of up to about 0.5 mg/vial; and (f) a water forinjection (WFI), wherein the water is degassed and deaerated with aninert gas.
 2. The composition of claim 1, wherein the DTPA-dextrancontains multiple DTPA groups conjugated to dextran in the molar rangeof about 2:1 to 12:1.
 3. The composition of claim 1, wherein theDTPA-dextran contains dextran in the average molecular weight range ofabout 5,000 to 20,000 Daltons.
 4. The composition of claim 1, whereinthe DTPA-dextran is DTPA-mannosyl-dextran containing a molar ratio rangeof about 2:1 to 12:1 conjugated Mannose groups to DTPA-dextran.
 5. Thecomposition of claim 1, wherein the non-reducing disaccharide isα,α-Trehalose Dihydrate.
 6. The composition of claim 1, wherein thenon-sulfhydryl anti-oxidant is L(+)-Ascorbic Acid Sodium salt.
 7. Thecomposition of claim 1, wherein the stannous salt is Stannous ChlorideDihydrate.
 8. The composition of claim 1, wherein the pH buffer andtranschelator is Glycine.
 9. The composition of claim 1, wherein theinert gas used to deaerated WFI is nitrogen.
 10. A method forstabilizing a DTPA-dextran cold kit for long-term storage, compromisingthe steps of: (a) adding an aqueous composition, compromising: (i) asugar selected from the group of non-reducing disaccharides with aconcentration up to 2% (w/v); (ii) a pH buffer selected from a group ofpH buffers in concentration range of up to about 0.5 mg/vial; to avessel containing about 90% of its target volume of degassed anddeaerated water for injection; (b) adding a non-sulfhydryl anti-oxidantwherein the concentration is in the range of about 0.5 mg/vial; (c)adjusting the solution pH to a target pH of 3.2±0.2 with 6 Nhydrochloric acid, while maintaining an inert gas sparge; (d) adding astannous salt wherein the concentration of the dihydrate form of thestannous salt is up to 75 micrograms/vial; (e) adding a DTPA-dextranwith a concentration of up to 0.50 mg/vial; (f) adjusting the solutionpH to a target pH of 3.2±0.2 with 6 N hydrochloric acid, whilemaintaining an inert gas sparge; (g) adjusting the volume of theformulation to 100% of its target volume with degassed and deaeratedwater for injection; (h) filtering the aqueous composition through a0.22 micron filter and filling the aqueous composition in to glass vialswith 1.0 mL±10% and placing stoppers in the neck of the vials; (i)removing the majority of the water content of the product, decreasingthe residual moisture to about less than 1% water content bylyophilization in step a; (j) backfilling the lyophilized product withan inert gas to about 11.5 p.s.i. prior to stoppering the vials in stepb; (k) crimping the lyophilized product vials with aluminum seals instep c; and (l) storing the crimped-sealed lyophilized product vials instep d at either 2° to 8° C., or 25° C.
 11. The method of claim 10,wherein the non-reducing disaccharide is α,α-Trehalose Dihydrate. 12.The method of claim 10, wherein the pH buffer and transchelator isGlycine.
 13. The method of claim 10, wherein the non-sulfhydrylanti-oxidant is L(+)-Ascorbic Acid Sodium salt.
 14. The method of claim10, wherein the stannous salt is Stannous Chloride Dihydrate.
 15. Themethod of claim 10, wherein the DTPA-dextran contains multiple DTPAgroups conjugated to dextran in the molar ratio range of about 2:1 to12:1.
 16. The method of claim 10, wherein the DTPA-dextran containsdextran in the average molecular weight range of about 5,000 to 20,000Daltons.
 17. The method of claim 10, wherein the DTPA-mannosyl-dextrancontaining a molar ratio range of about 2:1 to 12:1 conjugated mannosegroups to DTPA-dextran.
 18. A method for radiolabeling a DTPA-dextrancold kit with a Sodium ^(99m)Tc-Pertechnetate solution and a diluent foruse as a diagnostic radiopharmaceutical and for adjusting the finalsolution pH for patent comfort, compromising the steps of: (a) adding anaqueous Sodium Pertechnetate (Tc^(99m)) composition, compromising; (i)greater than about 100 Curies of Sodium Pertechnetate/mmol DTPA-dextranin a volume of 0.7 mL; (ii) allowing for a Tc^(99m) dose range fromabout 0.3 to 5.0 millicuries Tc^(99m) in a 0.2 mL dose in a final volumeof 1.0 mL; (b) adding an aqueous buffered saline diluent composition ina volume of 0.3 ml, compromising; (i) 0.5% (w/v) Sodium Chloride, USP;(ii) 0.107% Sodium Phosphate Heptahydrate; (iii) 0.036% PotassiumPhosphate; (iv) 0.4% Phenol; (v) adding water for injection (WFI) toq.s. to a final volume; and (vi) adding concentrated Sodium Hydroxide orHydrochloric Acid, as needed to adjust pH to about pH 7.0.